The present invention relates to nucleotide sequences for regulating gene expression in plant trichomes and, more particularly, to methods of utilizing such nucleotide sequences for synthesizing polypeptides and molecules of interest in plant trichomes.
Polypeptides can be expressed in a wide variety of cellular hosts. For economic reasons, genetically engineered unicellular microorganisms are most widely used for commercial production of polypeptides. However, in some cases, expression of mammalian proteins in unicellular organisms results in incorrect folding and processing of the expressed polypeptides leading to loss of biological or physiological activity of the obtained polypeptide. For these reasons, attempts have been made, with varying degrees of success, to express mammalian polypeptides in plants.
Transgenic plants are fast becoming a preferred system for the expression of many recombinant proteins, especially those intended for therapeutic purposes. One advantage of using plants is the potential for protein production on an agricultural scale at an extremely competitive cost. Among other advantages is that most plant transformation techniques result in a stable integration of the foreign DNA into the plant genome, so genetic recombination by crossing of transgenic plants is a simple method for introducing new genes, accumulating multiple genes into plants and avoiding the contamination of pathogens such as viruses and prions, which may affect human and animals. Furthermore, the processing and assembly of recombinant proteins in plants may also complement that in mammalian cells, which may be an advantage over the more commonly used microbial expression systems.
Although plants provide a suitable alternative to unicellular expression systems, several disadvantages characterize current approaches for production of protein in plants. First, the concentration of the produced protein is typically low (around 1% of total proteins) making pufification extremely difficult. Second, other compounds may interfere with protein purification or even damage the proteins during purification. Third, expressing foreign proteins in propagated plants can lead to environmental contamination and health risks associated with unwanted production of those proteins in cross pollinated plants.
In efforts of overcoming the above described limitations and while reducing the present invention to practice, the present inventors have discovered that plant trichomes enable compartmentalized production of foreign proteins as well as enzymatic production of novel chemicals, since many types of chemicals are naturally produced and even secreted from trichomes.
The above ground surfaces of many plants are covered with trichomes or hairs. The morphology of these structures can vary greatly with tissue type and species. Indeed, the botanical literature contains more than 300 descriptions (uniseriate, capitate-sessile, etc.) of various morphological types of such hairs (3, and references therein). Functionally, trichomes may be simple hairs that deter herbivores, guide the path of pollinators, or affect photosynthesis, leaf temperature, or water loss through increased light reflectance as in desert species. Alternatively, they may be more specialized tissues (glandular secreting trichomes) whose principal function(s) may be to produce pest- or pollinator-interactive compounds that are stored or volatilized at the plant surface. It has been suggested that in some desert species the principal role of glandular secreting trichomes is to produce such high levels of exudate that it forms a continuous layer on the plant surface. This layer may increase light reflectance and thereby reduce leaf temperature (30).
Trichomes develop projections from protodermal cells. Their structures arise from a series of anticlinal and periclinal divisions to form supporting auxiliary cells and glands. The appearance of glands atop supporting cells and the occurrence of exudate around gland cells has suggested that secretions are produced in gland cells and not by other epidermal or subepidermal cells.
In several species, such as tomato and potato, a unique type of trichomes accumulates certain protein (polyphenol oxidase) and compound (polyphenol) in the associated glands on the top of the trichome. When an insect lands on a leaf surface and contacts these trichomes, they discharge their inner compounds thereby contacting the insect and smearing it with a brown sticky compound, which is the product of enzymatic oxidation of the polyphenols (reviewed in 4).
The mass production, accumulation, and secretion of such proteins and chemicals involve a specific genetic mechanism. This genetic mechanism includes genes (5, 6) and promoters (7, 8, 9) acting in trichome cells and cells organelles suited for accumulation and secretion of mass products. This genetic mechanism allows, for example, trichome exudates to reach 16% of total dry weight of leaves of a certain tobacco species (10) and a single protein to reach 60% of total proteins or a concentration of 14 mg/mL in the trichome content of a solanum species (11, 12). The use of this genetic mechanism was suggested for tissue specific production and accumulation of natural and heterologous proteins as well as chemicals (6). New compounds produced can be beneficial for the plant itself by increasing resistance against pests such as insects, bacteria, and fungi (6), or for Molecular Farming or Bio-Farming of human or mammalian proteins for the use as therapeutics. In the latter, harvesting the proteins produced in the trichomes can be mechanized.
Directing protein expression into trichome cells may involve the use of polynucleotides originated form different origins. A candidate source for such regulatory elements is cotton as its fiber tissue is structurally modified trichomes. The promoter sequences of cotton fiber specific genes were shown to direct β-glucuronidase (GUS) expression to the trichome cells of tobacco plants (7, 9). Alteration of trichomes structure or chemistry by, for example, increasing cotton trichome length or by producing pigments in the fiber could be beneficial for the cotton industry.
Natural chemicals of trichomes are already used as flavor, aroma, medicinals, pesticides, and cosmetic ingredients (13, 14). Natural chemicals content was altered using antisense and co-suppression methods (6). However, enzymatic modifications of trichomes compounds via genetic engineering of genes, designed to produce other useful compounds in trichomes, was never shown before.
Several limitations had narrowed so far the use of plant trichomes for commercially production of heterologous proteins and novel chemicals.
First, protein yield is very limited in trichome cells and to date there is no existing method that enables commercially significant production of proteins in these cells. Although there are known promoter sequences that are capable of directing protein synthesis in trichomes (7, 8, 9), proteins expressed therefrom accumulated at average levels of accumulation of a single trichome protein and thus these promoters cannot be considered commercially useful for protein production, as is. Second, trichomes usually produce a mix of several metabolites, some of which (e.g., phenols and alkaloids), can inhibit protein accumulation or substantially hinder purification of desired compounds produced in trichomes (See material and methods in 12). Thus, reducing the levels of such harmful metabolites is required in order to improve harvesting and collection of the desired products. Third, the production of novel compounds in plants always involves risks of escape of genetic material (pollen and seeds) to the environment with potential damage to other organisms (plants, insects animals, human). Hence, when producing novel compounds one should consider the elimination of the possible spread of the new genetic material.
There is thus a widely recognized need for and it would be highly advantageous to have nucleotide sequences for regulating gene expression in plant trichomes methods of utilizing such nucleotide sequences for generating molecules of interest in plant trichomes.